Structural Studies of the Atypical Rio2 Kinase and N-Acetylglutamate Synthase

Thumbnail Image


Publication or External Link






Rio2 is required for small subunit ribosomal RNA (rRNA) maturation. It is specifically involved in site D cleavage of the 20S rRNA to produce the mature 18S rRNA. Loss of Rio2p is lethal and a decrease of Rio2p activity results in 20S accumulation in the cytoplasm. One of the goals of this thesis was to crystallize Rio2 from a eukaryotic organism in order to determine the structural differences between eukaryotic and archaeal versions. Another goal was to define the importance of individual domains. Yeast Rio2 was the first eukaryotic protein purified, but it formed only microcrystals. Human Rio2 was purified next, but the solubility was too low to set up crystal trays. Finally, Rio2 from Chaeotomium thermophilum was purified and crystals were obtained. The structure revealed a possibly inhibitory alpha helix blocking the active site. The role of the N-terminal winged helix domain of Rio2 in yeast was investigated and found not to be necessary for binding of Rio2p to the ribosome.

The crystal structure of the first N-acetylglutamate synthase (NAGS) was also determined. The crystal structure of NAGS complexed with acetyl-CoA and with CoA plus N-acetylglutamate was determined at 2.5 and 2.6-Å resolution. Each NAGS consists of an N-terminal amino acid kinase domain (AAK) domain and a C-terminal N-acetlyltransferase (NAT) domain connected by three amino acids. The monomers form a six membered ring with a trimer of dimers symmetry. The AAK domains form two dimeric contacts with other AAK domains. Each AAK domain interacts with the NAGS domain of another monomer at the polar ends of the ring. The NAGS domain contains the active site. The AAK domain is believed to bind arginine and also helps to bind acetyl-CoA. Structural insights suggest a one step mechanism in which both substrates bind and the acetyl group is directly passed from acetyl-CoA to the alpha amino group of gluatamate.

In addition, collaborative work on the structural characterization of the MphR(A) protein is reported.